CN-GELFrEE - Clear native gel-eluted liquid fraction entrapment electrophoresis

Rafael D. Melani, Henrique S. Seckler, Owen S. Skinner, Luis H.F. Do Vale, Adam D. Catherman, Pierre C. Havugimana, Marcelo Valle de Sousa, Gilberto B. Domont, Neil L. Kelleher, Philip D. Compton*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDSPAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

Original languageEnglish (US)
Article numbere53597
JournalJournal of Visualized Experiments
Volume2016
Issue number108
DOIs
StatePublished - Feb 29 2016

Keywords

  • Biomolecular assemblies
  • Chemistry
  • Clear native electrophoresis
  • Gel-eluted liquid fraction entrapment electrophoresis
  • Issue 108
  • Native fractionation
  • Polyacrylamide gel electrophoresis
  • Protein complexes

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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